The present invention relates to cells having mutated hERG gene potassium channels (Kv11.1) that are constitutively open, specific mutations that result in constitutively open Kv11.1 channels, and the potential uses for cells having constitutively open Kv11.1 channels in the development of improved drug screening assays.
The human ether-a-go-go related gene (hERG) encodes the Kv11.1 protein α-subunits that underlie the rapidly activating delayed rectifier K+ current (IKr) in the heart. The α-subunits combine to form the Kv11.1 potassium channels, which function in cardiac myocytes as voltage-gated channels to restore resting membrane potential after action potential generation.
Drug-induced acquired long QT syndrome (LQTS) has become an important liability for clinically available drugs and developmental compounds. LQTS is a heart condition associated with prolongation of repolarization following depolarization of the cardiac ventricles. It is associated with fainting and sudden death due to ventricular arrhythmias.
The mechanism commonly proposed for drug-induced QT interval prolongation is direct block of Kv11.1 channels or its native current, IKr. The drugs bind to a structurally unique receptor domain in the pore-S6 region of the channel to suppress K+ ion permeation. Drug binding (block) usually occurs preferentially to the “open state” (ion conducting state) of the channels. Screening for unanticipated Kv11.1 channel block is now expected of all new drugs submitted for review to the FDA and to other regulatory agencies worldwide, and Kv11.1 channel block screening is commonly now done within the pharmaceutical industry early in the life-cycle of new compound development. In recent years, Kv11.1 channel block by drugs has become one of the most common reasons for drug withdrawal from the marketplace and for the termination of lead compounds in development.
Under normal conditions, Kv11.1 channels are in a closed or rested state, where drugs bind to the ion channel with low affinity. During cell depolarization the Kv11.1 channels become active (open) to conduct K+ ions, and drugs can access and bind to a drug binding domain located in the channel pore-S6 region of the Kv11.1 protein. Drugs bind with high affinity to this open state, thus this open state is key in studying drug binding (block) efficacy to the channel. Kv11.1 channels also can inactivate (cease conducting ions) and when cells repolarize the Kv11.1 channel again reopens before returning to the rested or closed state.
In the Kv11.1 channel protein the S4 transmembrane spanning segment, as with most ion channel proteins, contains multiple charged amino acids and is thought to be the voltage-sensor that responds to changes in the transmembrane voltage resulting in the channel pore opening to conduct K+ ions. The S1-S3 transmembrane spanning segments are less well studied, but also contain charged amino acid residues and these segments are thought to modify channel gating properties.
We have described below mutations in the transmembrane spanning segments of the Kv11.1 channel protein that unexpectedly cause the channel to remain open at all times. The example below shows that these constitutively open mutant Kv11.1 channels are blocked by channel-blocking drugs. Thus, the mutant Kv11.1 channels of the present invention could provide a more efficient and effective assay for studying the tendency of drugs and other compounds to bind and block the Kv11.1 channels and for assessing the risk for drug-induced LQTS.